Eukaryotic genes contain introns which interrupt the continuity of the genetic information. Introns are removed from an RNA transcript of the gene by RNA splicing. This project continues an investigation of the mechanism of splicing of two kinds of RNA splicing in yeast: tRNA and mRNA. tRNA precursors are spliced in a two-step reaction. An endonuclease removes the intron and a ligase, requiring ATP, joins the exons together. The endonuclease will be purified to homogeneity. The mechanism by which it recognizes and cleaves the precursor will be studied. The ligase has already been purified to homogeneity. Further studies will explore its domain structure and the mechanism of the ligase reaction. Studies on a synthetic tRNA-Phe precursor will elucidate the role of secondary and tertiary structure of the mature domain in substrate recognition. mRNA precursors are spliced on a large particle called the spliceosome. Yeast mutants have been isolated called rna2-11 which define components of the spliceosome. The RNA gene products will be purified using an in vitro complementation assay. These products, once purified, can be used to define and characterize other RNA and protein components of the spliceosome. The spliceosome will be purified and characterized by electron microscopy and biochemical fractionation. The pathway by which it is assembled and disassembled will be determined. A possible role of hnRNPs in mRNA splicing will also be investigated.